Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is a fatal condition occurring in midlife where motor neurons degenerate in the spinal cord and motor cortex. One genetic defect in familial ALS (FALS) has now been linked to Sod 1, the gene that encodes the cytosolic CuZnSOD enzyme. Postulated mechanisms for onset and progression of FALS include gain-in-function of Sod 1, i.e., increased peroxidase or hydroxyl radical activity. ESR spin-trapping with oxygen-17 hydrogen peroxide was used to investigate the mechanism of hydroxyl radical adduct of DMPO formation from the SOD and SOD mutants. The relative ratios of oxygen-17 hydroxyl radical adduct of DMPO formed in the Fenton reaction were 90% and 10%, respectively. The reaction of the wild type SOD (WT-SOD) with oxygen-17 hydrogen peroxide in bicarbonate/carbon dioxide buffer yielded 56 +/- 0.47% of oxygen-17 hydroxyl radical adduct of DMPO spin adduct and 44 +/- 0.47% oxygen-16 hydroxyl radical adduct of DMPO. Similar results were obtained from the reaction between FALS SOD mutants and oxygen-17 hydrogen peroxide: oxygen-17 hydroxyl radical adduct of DMPO, 57 +/-1.41%, and oxygen-16 hydroxyl radical adduct of DMPO, 43 +/- 1.41% from A4V and 55 +/- 0.82% oxygen-17 hydroxyl radical adduct of DMPO and 45 +/- 0.82% oxygen-16 hydroxyl radical adduct of DMPO from G93A. These results indicate that a significant fraction of hydroxyl radical adduct of DMPO formed during the reaction of SOD and FALS SOD mutants with hydrogen peroxide is derived from the incorporation of oxygen atom from water. We found no evidence for increased peroxidase or hydroxyl radical activity in ALS mutant SOD compared with the WT-SOD.[unreadable]